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J. Biol. Chem., Vol. 281, Issue 23, 15997-16005, June 9, 2006

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Activity-based Protein Profiling Implicates Urokinase Activation as a Key Step in Human Fibrosarcoma Intravasation^*

From the Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037

Entry of malignant cells into the vasculature (i.e. intravasation) requires proteolytic remodeling of the extracellular matrix so that tumor cells may pass through the local stroma and penetrate the vessel wall. The circulatory system then provides a means of transporting tumor cells to distant sites where they extravasate and establish metastatic lesions. This study utilizes activity-based protein profiling to compare the active serine hydrolase repertoire in high intravasating (HT-hi/diss) and low intravasating (HT-lo/diss) variants of the human fibrosarcoma HT-1080 cell line to determine which enzyme(s) play a role in intravasation. Activity-based protein profiling revealed multiple serine hydrolases with altered activity between HT-hi/diss and HT-lo/diss cells, with the largest difference being the activity of urokinase-type plasminogen activator (uPA). Levels of inactive uPA zymogen were similar between the two cell variants, but only HT-hi/diss conditioned medium contained active uPA, suggesting that uPA activation may contribute to the enhanced intravasation of HT-hi/diss cells. To analyze the role of uPA activity specifically in the process of intravasation, we grafted cells from the two HT-1080 variants onto the chorioallantoic membrane of chick embryos and measured levels of tumor cell intravasation in the distal chorioallantoic membrane using quantitative human-specific Alu PCR. Inhibition of uPA activity with natural (plasminogen activator inhibitor-1) or synthetic (amiloride) inhibitors diminished HT-hi/diss Matrigel invasion in vitro and intravasation and metastasis in vivo. Additionally, treatment of HT-lo/diss tumors with exogenous active uPA increased the number of intravasated cells in vivo. These results indicate that active uPA promotes tumor cell intravasation and that uPA activation appears to be a key step in tumor progression.

Received for publication, February 8, 2006 , and in revised form, April 10, 2006.

^* This work was supported by National Institutes of Health (NIH) Grants CA55852 and CA105412 (to J. P. Q.) and NIH Grant HL07695 (to M. A. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7190; Fax: 858-784-7333; E-mail: jquigley{at}scripps.edu.

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